Protein kinase C activators and their use in decreasing expression of cell antigens

ABSTRACT

A composition for the upregulation of expression of cell antigens, without inducing shedding, which comprises a protein kinase C activator is provided by this invention. Further provided by this invention is a method of detecting and treating tumor cells comprising contacting tumor cells with an effective amount of a protein kinase C activator for the upregulation of expression of antigens of tumor cells, without inducing antigen shedding, and detecting the presence of said antigen or then further contacting said tumor cells with an effective amount of an antibody directed to said antigen.

This is a national application based on PCT International ApplicationPCT/US94/10755, filed Sep. 21, 1994 which is a continuation-in-part ofU.S. Ser. No. 08/124,716 filed Sep. 21, 1993, now U.S. Pat. No.5,681,680, the contents of which are hereby incorporated in theirentirety.

The invention described herein was made in the course of work underGrant Nos. CA 35675 and CA 43208 from the National Institute ofHealth-National Cancer Institute. The United States Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byArabic number within brackets. Full citations for these publications maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed therein.

The expression of both histocompatibility antigens and tumor associatedantigens (TAAs) of tumor cells can be augmented by treatment withbioresponse modulators, such as interferon and tumor necrosis factor-α,and phorbol ester tumor promoters, such as TPA[1,13,14,16,18,22,23,27,28]. Upregulation of additional cellularantigens can be induced to a similar extent in both normal and tumorcells by bioresponse modulators indicating that this effect is a generalproperty of these compounds and not restricted to TAAs or cells of aspecific histotype (for review see [1,23]). For example, variousinterferons have been shown to enhance the expression ofhistocompatibility antigens, cellular antigens and TAAs in breastcarcinoma, central nervous system tumors, colon carcinoma and melanomacells [21-23,27,28,31,38]. In addition, when administered to animalscontaining human tumor xenografts, recombinant human interferon augmentsthe ability of excised tumors to bind monoclonal antibodies specific forTAAs [13,21,36,42]. The use of bioresponse modifiers for increasing theexpression of TAAs by tumor cells may prove useful in reducing theantigenic heterogeneity in tumors in vivo and augmenting the ability ofmonoclonal antibodies to bind to tumors (for review see [1,23,25,26]).

TPA and recombinant human leukocyte (IFN-α), fibroblast (IFN-β) andimmune (IFN-γ) interferons increase both the expression and shedding ofthe tumor associated antigen BCA 225 by T47D cells and MCF-7 humanbreast carcinoma cells [36]. These compounds also increase theexpression of the TAA carcinoembryonic antigen (CEA) and HLA Class II-DRantigen in both T47D and MCF-7 cells [20,36]. The mechanism by which TPAinduces its diversity of effects in target cells is believed to bemediated initially by its binding to the Ca²⁺-activated andphospholipid-dependent enzyme PKC which is the high affinity receptorfor TPA (for review see [10, 43,44]). As a consequence of activation ofPKC many important biochemical processes are initiated in target cells,including both positive and negative feedback controls in signaltransduction pathways (for review see [43,44]). Recent studies haveimplicated PKC activation in mediating both antiviral activity andspecific gene regulatory changes induced by IFN-α, IFN-β, and IFN-γ[6,8,37,46,47,50] and for review see [9]. The purpose of the presentstudy was to explore the possible relationship between PKC activationand antigen upregulation induced by phorbol esters and interferon. Withthis aim in mind applicants have determined the effect of the syntheticPKC-activator ADMB, the natural PKC activators TPA and MEZ and thecombination of PKC-activators and the PKC-inhibitor H-7 on the antigenicphenotype of T47D cells. To determine if similar biochemical pathwaysare involved in the ability of IFN-β and IFN-γ to alter the antigenicphenotype of T47D cells, applicants have also evaluated the effect ofH-7 on interferon upregulation of the same antigens in T47D cells.

The effect of a synthetic protein kinase C (PKC) activator3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB) andthe natural PKC-activating tumor promoting agents12-0-tetradecanoyl-phorbol-13-acetate (TPA) and mezerein (MEZ) on theantigenic phenotype of carcinoma cells was studied. All three agentsincreased the surface expression of the tumor associated antigen such asBCA 225 and also of various cellular antigens, including HLA Class IIantigens, intercellular adhesion molecule-1 (ICAM-1) and c-cerbB-2.Expression of the same antigens was also upregulated to various extentin T47D cells by recombinant fibroblast (IFN-β) and immune (IFN-γ)interferon. Shedding of BCA 225 from T47D cells was induced by TPA, MEZ,IFN-β and IFN-γ, whereas ADMB did not display this activity. The abilityof ADMB, TPA and MEZ to modulate the antigenic phenotype of T47D cellsappears to involve a PKC-mediated pathway, since the PKC inhibitor, H-7,eliminates antigenic modulation. In contrast, the ability of IFN-β andIFN-γ to enhance HLA Class II antigens, c-erbB-2 and ICAM-1 expression,was either unchanged or modestly reduced by simultaneous exposure toH-7. Analysis of steady-state mRNA levels for HLA Class I antigens, HLAClass II-DRβ antigen, ICAM-1, and c-erbB-2 indicated that the ability ofH-7 to inhibit expression of these antigens in TPA-, MEZ-, andADMB-treated cells was not a consequence of a reduction in thesteady-state levels of mRNAs for these antigens. The results of thepresent investigation indicate that the biochemical pathways mediatingenhanced antigenic expression in T47D cells induced by TPA, MEZ and thesynthetic PKC activator ADMB are different than those induced byrecombinant interferons. Furthermore, upregulation of antigenicexpression in T47D cells can occur by both a PKC-dependent or aPKC-independent pathway.

SUMMARY OF THE INVENTION

This invention provided a composition for the upregulation of expressionof all antigens without inducing shedding which comprises a proteinkinase C activation for the upregulation of expression of a cell antigenwithout inducing shedding of said antigen from the cell.

Further provided by this invention is a method for decreasing tumor cellheterogeneity by upregulating the expression of an antigen withoutinducing shedding of said antigen which comprises administering anamount of a protein kinase C activator to cells to induce upregulationof expression of an antigen without inducing shedding of said antigen.

Additionally, this invention provides a method of detecting tumor cellscomprising contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells, without inducing shedding, and detecting the presence ofsaid antigen.

A method for treating tumor cells is also provided. This methodcomprises contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells without inducing antigen shedding and then contacting saidtumor cells with an effective amount of an antibody directed to saidantigen.

This invention also provides a pharmaceutical composition forupregulating the expression of antigens without inducing antigenshedding which comprises a pharmaceutically acceptable carrier and aneffective amount of a protein kinase C activator.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Effect of H-7, TPA, MEZ and ADMB on T47D cell growth and DNAsynthesis. Cell growth assays are presented in (A). T47D cells wereseeded at 1×10⁴ cells/35 mm tissue culture place, the medium was changedwith the indicated compounds 24 hr later and cell numbers weredetermined after an additional 72 hr growth at 37° C. by CoulterCounter. Results are the average of triplicate samples/experimentalcondition which varied by ≦10%. DNA synthesis assays are presented in(B). T47 D cells were seeded at 1.25×10⁴ cells in 0.2 ml of media in 96microtiter plates. Every 24 hr, cultures received 1 μCi ofmethyl-³H-thymidine and 8 hr later cells were harvested and TCAprecipitable counts were determined. Results are the average fromreplicate samples exposed to the indicated compounds for 72 hr.Replicate samples varied by ≦10% and replicate studies varied by ≦15%.Further details can be found in “Detailed Description Of The Invention”.

FIG. 2: Effect of H-7 on the upregulation of HLA Class II antigens-andc-erbB-2 antigen expression in T47D cells induced by ADMB, TPA and MEZ,T47D cells were grown for 72 hr in the presence of 0.1 μg/ml of ADMB,TPA or MEZ, used alone or in combination with 0.1 kg/ml of H-7. Cellsurface antigen expression was then determined by FACS analysis asdescribed in “Detailed Description Of The Invention.” Base-line controlantigen expression is given the value of 1.0. The values presented arethe fold-change, which represents the ratio of the experimental MFIvalue versus the control MFI value for the specific antigen tested, influorescence in experimental versus control samples. The resultspresented are from a single experiment employing replicate samples.Similar results ≦15% have been obtained in two additional studies.Further details can be found in “Detailed Description Of The Invention.”

FIG. 3: Effect of interferon, ADMB, TPA and MEZ, alone and incombination with H-7, on steady-state mRNA levels of HLA Class Iantigens, HLA Class II-DR_(β) antigen, c-erbB-2, ICAM-1 and GAPDH inT47D human breast carcinoma cells. T47D cells were grown for 72 hr inthe absence (control) or presence of 500 units/ml of IFN-α or IFN-β, 50units/ml of IFN-γ, 0.1 μg/ml of TPA, MEZ or ADMB. Cultures were alsogrown in the presence of 0.1 μg/ml of H-7 for 72 hr with and without theadditional compounds indicated above. Total cytoplasmic RNA was isolatedand processed as described in “Detailed Description Of The Invention.”Control: CON; recombinant human leukocyte interferon-A: IFN-αA;recombinant human fibroblast interferon: IFN-β; recombinant human immuneinterferon: IFN-γ; ADMB;3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol; TPA;12-0-tetradecanoyl-phorbol-13-acetate; MEZ; mezerein; H-7:(1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition for the upregulation of expressionof cell antigens without inducing shedding which comprises a proteinkinase C activator for the upregulation of expression of a cell antigenwithout inducing shedding of said antigen from the cell. In oneembodiment of the invention the protein kinase C activator is asynthetic protein kinase C activator. In the preferred embodiment of theinvention the protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADME).

In an embodiment of the invention the cell antigen is a tumor associatedantigen (TAA). In yet another embodiment of the invention the cellantigen is a cell surface antigen. The cell antigen may also be ahistocompatibility antigen. Examples of tumor associated antigens thatcan be upregulated by this invention include, but are not limited to,BCA 225, c-erb B2, carcinoembryonic antigen or CA19.9. In one preferredembodiment of the invention the cell surface antigen is intercellularadhesion molecule-1.

The tumor of the tumor associated antigen may be, but is not limited to,breast carcinoma or colon carcinoma.

In an embodiment of the above-described composition, the tumor of thetumor associated antigen is a colon carcinoma. In a further embodiment,the colon carcinoma associated antigen is a histocompatibility antigen.In a still another embodiment, the histocompatibility antigen is ClassII HLA-DR. In another embodiment, the colon carcinoma associated antigenis CA19.9.

Further provided by this invention is a method for decreasing tumor cellheterogeneity by upregulating the expression of an antigen withoutinducing shedding of said antigen which comprises administering anamount of a protein kinase C activator to cells to induce upregulationof expression of an antigen without inducing shedding of said antigen.In one embodiment of the invention the protein kinase C activator is asynthetic protein kinase C activator. In a preferred embodiment of theinvention the synthetic protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB). Whenusing ADMB for decreasing tumor cell heterogeneity by upregulating theexpression of an antigen, without inducing shedding, the effectiveamount is from about 0.01 μg/ml to about 10 μg/ml.

In one embodiment of the invention the antigen is a tumor associatedantigen. In another embodiment of the invention the antigen is ahistocompatibility antigen. In yet another embodiment of the inventionthe antigen is a cell surface antigen. The tumor of the tumor associatedantigen can be, but is not limited to, breast carcinoma or coloncarcinoma. In certain embodiments of the invention the tumor associatedantigen is BCA 225. In other embodiments of the invention the tumorassociated antigen is carcinoembryonic antigen. In yet anotherembodiment of the invention the tumor associated antigen is c-erb B2. Inanother embodiment of the invention the cell surface antigen inintercellular adhesion molecule-1.

In a further embodiment, the colon carcinoma associated antigen is ahistocompatibility antigen. In a still another embodiment, thehistocompatibility antigen is Class II HLA-DR. In another embodiment,the colon carcinoma associated antigen is CA19.9.

This invention also provides a method of detecting tumor cellscomprising contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells, without inducing antigen shedding, and detecting thepresence of said antigen. In one embodiment of the invention the proteinkinase C activator is a synthetic protein kinase C activator. In thepreferred embodiment of the invention the synthetic protein kinase Cactivator is 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol(ADMB). When using ADMB in the subject invention the effective amount isfrom about 0.01 μg/ml to about 10 μg/ml. The tumor of the tumorassociated antigen may be, but is not limited to, breast carcinoma orcolon carcinoma. In certain embodiments of the invention the tumorassociated antigen is BCA 225. The tumor associated antigen can also becarcinoembryonic antigen or c-erb B2. In yet another embodiment of theinvention the antigen is intercellular adhesion molecule-1.

In a further embodiment, the colon carcinoma associated antigen is ahistocompatibility antigen. In a still another embodiment, thehistocompatibility antigen is Class II HLA-DR. In another embodiment,the colon carcinoma associated antigen is CA19.9.

Further provided by this invention is a method of treating tumor cellscomprising contacting tumor cells with an effective amount of a proteinkinase C activator for the upregulation of expression of antigens oftumor cells without inducing antigen shedding and then contacting saidtumor cells with an effective amount of an antibody directed to saidantigen. In one embodiment of the invention the protein kinase Cactivator is a synthetic protein kinase C activator. In the preferredembodiment of the invention the synthetic protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB). Whenusing ADMB, the effective amount is from about 0.1 μg/ml to about 10μg/ml. The antigen may be, but is not limited to, a tumor associatedantigen or a cell surface antigen. The tumor cells may be, but are notlimited to, breast carcinoma or colon carcinoma. In preferredembodiments of the invention the tumor associated antigen is BCA 225,carcinoembryonic antigen, or c-erb B2. In yet another embodiment of theinvention the cell surface antigen is intercellular adhesion molecule-1.

In a further embodiment, the colon carcinoma associated antigen is ahistocompatibility antigen. In a still another embodiment, thehistocompatibility antigen is Class II HLA-DR. In another embodiment,the colon carcinoma associated antigen is CA19.9.

Additionally, this invention provides a pharmaceutical composition forupregulating the expression of antigens without inducing antigenshedding which comprises a pharmaceutically acceptable carrier and aneffective amount of a protein kinase C activator. In one embodiment ofthe invention the protein kinase C activator is a synthetic proteinkinase C activator. In the preferred embodiment of the invention thesynthetic protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB). Whenthe protein kinase C activator is ADMB, the effective amount is fromabout 0.01 μg/ml to about 10 μg/ml. The antigen may be, but is notlimited to being, a tumor associated antigen, a cell surface antigen, orhistocompatibility antigen. The tumor of the tumor associated antigenmay be, but is not limited to, breast carcinoma or colon carcinoma. Inone embodiment of the invention the tumor associated antigen is ECA 225.In another embodiment of the invention the tumor associated antigen iscarcinoembryonic antigen. In yet another embodiment of the invention thetumor associated antigen is c-erb B2. In one embodiment of the inventionthe cell surface antigen is intercellular adhesion molecule-1.

In a further embodiment, the colon carcinoma associated antigen is ahistocompatibility antigen. In a still another embodiment, thehistocompatibility antigen is Class II HLA-DR. In another embodiment,the colon carcinoma associated antigen is CA19.9.

For the purposes of this invention, “physiologically acceptable carrier”means any of the standard pharmaceutical carriers. Examples of suitablecarriers are well known in the art and may include, but are not limitedto, any of the standard pharmaceutical carriers.

Methods of determining the effective amounts are well known in the art.A person of ordinary skill in the art can easily extrapolate theeffective amounts as determined in vitro, and apply it to livingorganisms to determine the effective concentrations in vivo.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Materials and Methods Cell Cultures

The T47D clone 11 human breast carcinoma cell line [32] was grown inRPMI 1640 medium supplemented with 2mM L-glutamine, 1 mM sodiumpyruvate, fungizone (0.25 μg/ml), streptomycin (50 μg/ml), penicillin(50 U/ml), 10% fetal bovine serum (FBS), β-estradiol and insulin (0.1μu/ml) at 37° C. in a 5% CO₂/95% air humidified incubator. Cultures weremaintained in the logarithmic phase of growth by subculturing at a 1:5or 1:10 split-ratio when cells approached confluency.

Growth and ³H-Thymidine Incorporation Assays

T47D cells were seeded at 5×10³ cells/ml in 35 mm tissue culture platesand 24 hr later the medium was changed with the indicated compounds.Seventy-two hr later the cells were resuspended in trysin/versene(0.125%/0.02%, w/w) and counted using a Z_(M) Coulter Counter (CoulterElectronics). For ³H-thymidine incorporation studies, T47D cells wereseeded at 1.25×10⁴ cells in 0.2 ml of media in 96 well microtiterplates. Every 24 hr, plates received 1 μCi of methyl-³H-thymidine(specific activity 10 μCi/mmol) (ICN Radiochemicals, Irvine, Calif.),cells were harvested 8 hr after the addition of labeled thymidine andTCA precipitable counts [55] were determined using a Packardscintillation counter. Replicate samples varied by ≦10% and replicatestudies varied by ≦15%.

Monoclonal Antibodies

IgG₁ murine monoclonal antibody Cu18 and Cu46 recognize two differentepitopes of a highly restricted breast carcinoma associated glycoproteinof M_(r) 225,000 to 250,000 (BCA 225) [41]. This TAA is expressedintracytoplasmically and on the membrane of 94% of breast tumors testedand in the T47D human breast carcinoma cell line. BCA 225 is shed intothe culture medium by T47D cells and into the sera of breast cancerpatients. IgG_(2a) monoclonal antibody L243 (anti HLA Class II-DR)recognizes a monomorphic HLA Class II-DR-α epitope (ATCC M355).Monoclonal antibody CL203.4 [40], which recognizes ICAM-I, was kindlyprovided by Dr. S. Ferrone, New York Medical College, NY. The c-erbB-2monoclonal antibody which recognizes the extracellular domain ofc-erbB-2 was obtained from Triton Biosciences, Inc., Alamedia, Calif.Monoclonal antibodies Cu18 and Cu46 were used at 0.05 μg/ml andmonoclonal antibodies interacting with HLA Class II-DR, ICAM-1 andc-erbB-2 were used at 10 μg/ml. For each experiment, isotype matchedcontrol backgrounds (IgG for Cu18, Cu46 and ICAM-1, IgG_(2a) for HLAClass Ii and IgG_(2b) for c-erbB-2) were subtracted from theexperimental results. Background from antimouse IgG FITC antibody wasalso subtracted from experimental results. Applicants never observedbackgrounds higher than 2% of total cells for isotypic mouse IgG orhigher than 5% for antimouse IgG FITC.

Analysis of TAA and Cellular Antigen Expression by FluorescenceActivated Cell Sorter (FACS) Analysis

T47D cells treated with the various compounds were analyzed by flowcytometry using appropriate monoclonal antibody concentrations asdescribed previously [36]. Briefly, 1×10⁵ cells were incubated with thetest antibody for 30 min at 4° C., washed 3× with PBS and incubated witha goat α mouse F (ab)₂ FITC conjugated test antibody at a 1:40 dilutionfor 30 min at 4° C. Cells were then washed 3× with PBS and analyzed on aFACStar (Beckon Dickinson, Mountain View, Calif.) or a Coulter Epics IVFACS (Coulter, Hialeah, Fla.). Results are expressed as meanfluorescence intensity (MFI) units which were determined as describedpreviously [36]. MFI=(mean channel fluorescence in fluorescence positiveantibody−binding cells×% of fluorescence positive antibody−bindingcells)−(mean channel fluorescence in unstained cells×% of fluorescencepositive cells in the unstained population). All studies were performeda minimum of three or four times with duplicate samples in eachexperiment. Replicate samples within individual experiments varied by≦10% and variation between experiments were generally ≦20%.

Analysis of the Synthesis and Shedding of BCA 225

After appropriate incubation times with the various compounds celllysates were prepared from T47D cells. Cells were washed 3× in PBS, pH7.6, pelleted and incubated for lhr at room temperature in 20 mM TrisHCl, pH 7.4, with PMSF. Cultures were then homogenized with a Teflonhomogenizer, centrifuged at 3000 RPM for 10 min at 4° C. and thesupernatant was mixed 1:2 with 20 mM Tris-HCl, pH 7.4, containing 0.5%NP40 (Sigma). After 1 hr at 4° C., the mixture was spun at 3000 RPM for10 min and the supernatant was passed through an EXTRACTIGEL column(Pierce, Ill.) to remove excess detergent. Protein concentration wasdetermined by the BCA micro-method (Pierce, Ill.). BCA 225 levels incell extracts and supernatants from control and treated T47D cells werequantitated using a double-determinant ELISA assay [4,36]. Briefly, Cu18monoclonal antibody coated Nunc Immunoplates (Nunc, Denmark) wereblocked with 1% BSA (Sigma, RIA Reagent Grade) and incubated with a 1:2dilution of supernatant in duplicate. Standard values for a partiallypurified BCA 225 preparation were used at a range of 0 to 10 μg/ml inRPMI 1640 plus 10% FBS (T47D growth medium). After 2 hr incubation andthree washings with PBS 0.1% Tween 20 a Cu46 monoclonal antibodyconjugated to peroxidase was applied to the plate, incubated for 2 hr,washed 6× in PBS 0.1% Tween 20 and the reaction was developed with 16 ngOPD (Sigma, St. Louis) and 4 μl of 30% H₂O₂ in McIlvans buffer, pH 9.6.The plates were read on a Dynatech Elisa reader and a linear standardcurve was generated and used to calculate the relative amount of BCA 225in cell lysates and shed into the culture medium. Results were adjustedto ng BCA 225 per mg of protein, or per 1×10⁶ cells. Replicate samplesvaried by ≦10% and replicate experiments varied by ≦20%.

RNA Isolation and Northern Blotting Analysis

Steady-state levels of HLA Class I, HLA Class II-DR_(β), c-erbB-2 andICAM-I mRNA in control and treated T47D cells were determined byNorthern blotting analysis of total cytoplasmic RNA probed withappropriate ³²P-labeled gene probes as previously described [3,56].Northern blots were also probed with a ³²P-labeled glyceraldehydephosphate dehydrogenase (GAPDH) [12,54] gene probe to verify equal mRNAexpression under various experimental conditions. Followinghybridization, the filters were washed and exposed for autoradiography.Radioautograms were analyzed by densitometer to determine fold-change inmRNA expression as a result of treatment with the different antigenicmodulating agents, with or without cocultivation with H-7.

Reagents

Recombinant human leukocyte (IFN-αA) and immune (IFN-γ) interferons wereproduced in Escherichia coli and purified to homogeneity as previouslydescribed. [19,49,53]. These interferons were kindly provided by Dr.Sidney Pestka, UMDNJ-Robert Wood Johnson Medical School, Piscataway,N.J. Recombinant human fibroblast (IFN-β) interferon, with a serinesubstituted for a cysteine at position 17 of the molecule [39], wassupplied by Triton Biosciences Inc., Alameda, Calif. as a lycophilizedpowder with a concentration of 4.5×10⁷ units/ml. The interferon titerswere determined by a cytopathic effect inhibition assay with vesicularstomatitis virus on a bovine kidney cell line (MDBK) or human fibroblastAG-1732 cells[49]. Concentrated stocks of interferons were aliquots,frozen at −80° C., thawed immediately prior to use and diluted to theappropriate concentrations in DMEM supplemented with 5 or 10% FBS. TPA(12-0-tetradecanoyl-phorbol-13-acetate), MEZ (mezerein), and ADMB(3-(N-Acetylamino)-5-(N-Decyl-N-Methylamino)-benzyl alcohol) wereobtained from LC Services Corp., Woburn, Mass. Stock solutions of 1mg/ml (TPA and MEZ) or 10 mg/ml (ADMB) were prepared indimethylsulfoxide, aliquoted and stored at −20° C. The PKC inhibitor H-7(1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) (Hidakaet al., 1984) (Sigma) was prepared in distilled H₂O and stored at 4° C.For experiments, aliquots were thawed and dispensed in Dulbecco'smodified Eagle's medium (DMEM) containing 5 or 10% FBS to yieldappropriate final concentrations. The solvent DMSO at (0.025 to 0.05%)did not alter the growth or antigenic expression of T47D cells.

EXPERIMENTAL RESULTS Effect of TPA, MEZ and ADMB on Growth and DNASynthesis in T47D Cells

In preliminary studies, the dose-range and time-course of induction ofantigenic enhancement in T47D cells by TPA, MEZ and ADMB, as well as theeffect of varying doses of H-7 on this process, was determined (data notshown). These studies indicated that the optimum effect on antigenicexpression in T47D cells exposed to TPA, MEZ or ADMB occurred by 72 hr.The most effective dose of TPA, MEZ or ADMB inducing upregulation of BCA225, HLA Class II antigens, ICAM-1 and c-erbB-2 in T47D cells was foundto be 0.1 μg/ml. The ability of these PKC activators to induce increasedantigenic expression in T47D cells was inhibited by the simultaneousexposure to 0.1 μg/ml of the PKC inhibitor H-7 (Tables 1 and 2). Theeffect of TPA, MEZ, ADMB and H-7 on 72 hr growth and ³H-thymidineincorporation in T47D cells is shown in FIG. 1. When exposed to 0.1μg/ml of the respective compounds, growth and DNA synthesis wassuppressed to the greatest degree in TPA treated cells. In contrast, atthe same dose of 0.1 μg/ml, MEZ and ADMB only marginally altered growthand DNA synthesis in T47D cells (FIG. 1). No additive or synergisticeffect on 72 hr growth suppression was observed when TPA or MEZ wereused in combination with 0.1 μg/ml of H-7 (data not shown).

Effect of TPA, MEZ and ADMB, Alone and in Combination with H-7, on theAntigenic Phenotype of T47D Cells

When tested for reactivity with specific monoclonal antibodies, controlT47D cells displayed the following constitutive antigenic phenotype: 10to 20% of cells (with a mean channel fluorescence of 180 to 210) werepositive for HLA Class II antigen (HLA-DR) expression; 5 to 10% (with amean channel fluorescence of 110-130) were positive for c-erbB-2expression; 80 to 90% (with a mean channel fluorescence of 170 to 200)were positive for ICAM-1 expression; 85 to 95% (with a mean channelfluorescence of 180 to 210) were positive for the TAA BCA 225 (asmonitored by the monoclonal antibody Cu18); and 60 to 70% (with a meanchannel fluorescence of 140 to 170) were positive for BCA 225 (asmonitored by the monoclonal antibody Cu46). The effect of TPA and MEZ,alone and in combination with 0.1 μg/ml of H-7, on HLA Class IIantigens, c-erbB-2 and ICAM-1 expression in T47D cells is shown inTable 1. H-7 did not significantly alter the de novo expression of anyof these antigens in T47D cells. However, when administered inconjunction with TPA or MEZ, H-7 effectively blocked the ability ofthese PKC stimulators to enhance antigenic expression. In the experimentshown in Table 1, MEZ was somewhat more effective than TPA in enhancingc-erbB-2 and ICAM-1 expression. An increased activity, at comparabledoses, of MEZ over TPA in enhancing the expression of these antigens, aswell as the TAA BCA 225, has been found in several additional studies(unpublished data and Table 2 and FIG. 2). For comparison purposes, asingle experiment is shown in Table 1. In this experiment, thecombination of MEZ+H-7 resulted in the lack of detectable c-erbB-2expression. This result may reflect technical difficulties rather than acomplete suppression in c-erbB-2 expression, since in additional studiesH-7 blocked the ability of MEZ to enhance the expression of this antigenwithout completely eliminating c-erbB-2 expression (unpublished data andFIG. 2).

Recent computer modeling studies have resulted in the synthesis ofcompounds which inhibit the binding of phorbol esters to PKC and whichactivate PKC in platelets resulting in the phosphorylation of a specific40 kDa protein substrate [58]. Applicants have presently tested one ofthese phorbol ester pharmacophores, ADMB, for its ability to upregulatethe same antigens in T47D cells previously shown to be modulated by TPAand MEZ. A comparison of the efficacy of upregulation of HLA Class IIantigens and c-erbB-2 in T47D cells exposed to TPA, MEZ and ADMB, in thepresence or absence of H-7, is presented in FIG. 2. In the case of HLAClass II antigens, ADMB was somewhat more effective than TPA and MEZ ininducing upregulations, whereas H-7 reduced or eliminated enhancementwhen applied in combination with these PKC activators. In the case ofc-erbB-2, MEZ was the most effective PKC activator tested in enhancingexpression and as observed with HLA Class II antigens H-7 reduced thisantigenic upregulation.

A series of experiments were conducted to determine the effect of TPA,MEZ and ADMB, alone and in combination with H-7, on the synthesis,surface expression and shedding of the TAA BCA 225 (Table 2). Thesynthesis of BCA 225 was increased following exposure to all of the PKCactivators, with MEZ being most effective in enhancing the synthesis ofthis TAA. Similarly, MEZ was the most effective of the three PKCactivators in enhancing the surface expression of BCA 225 in T47D cells.As observed with the other antigens analyzed, H-7 effectively blockedboth the enhanced synthesis and surface expression of BCA 225. Whencompared for their ability to induce shedding of BCA 225 from T47Dcells, a differential response was observed between the three PKCactivators (Table 2). Both MEZ and TPA enhance shedding of BCA 225, withMEZ again being more effective than TPA, whereas ADMB did not inducethis effect in T47D cells. As observed with both synthesis and shedding,H-7 reduced the ability of MEZ and TPA to induce shedding of BCA 225.

Effect of IFN-β and IFN-γ, Alone and in Combination with H-7, on theAntigenic Phenotype of T47D Cells

Applicants have previously demonstrated that both IFN-β and IFN-γ caneffectively enhance the expression of BCA 225, HLA Class II antigens andICAM-I expression in T47D cells [36]. Optimum enhancement was observedby 72 hr with ranges of interferon of 500 to 1000 units/ml of IFN-α orIFN-β and 50 to 500 units/ml of IFN-γ, in the presence or absence of 1.0μg/ml of H-7, is shown in Tables 3 and 4. A higher dose of H-7 wasemployed in this study because the lower H-7 concentration of 0.1 μg/mldid not block enhanced antigenic expression in interferon treated T47Dcells (data not shown). IFN-γ was more effective (even at lowerconcentrations) than IFN-β in enhancing the expression of BCA 225, HLAClass II antigens and ICAM-1. IFN-α also increased the expression of thethree antigens tested, but to a lower extent than IFN-β or IFN-γ (datanot shown). For example, in the experiment shown in Table 3, 500units/ml of IFN-α enhanced HLA Class II expression from an MFI of 2,814to an MFI of 11,660 (a 4-fold increase) as opposed to a 11- and a69-fold increase, respectively, in HLA Class II expression in cellsexposed to IFN-β or IFN-γ (data not shown). In a number of experiments,the de novo level of expression of specific antigens and the absolutelevel of upregulation varied. Part of this difference may reflect theuse of a different FACS with different sensitivities for determining MFIunits and/or innate differences in antigenic expression of cells as aconsequence of the cell cycle. Although this makes it difficult todirectly compare absolute levels of upregulation with the phorbol estersand ADMB versus the interferons, it still permits a comparison of theeffect of H-7 on upregulation. In experiments simultaneously comparingthe various compounds, IFN-γ was generally a more effective enhancer ofHLA Class II antigens and ICAM-1 than the other agents, whereas MEZ wasgenerally more effective in modifying c-erbB-2 and BCA 225 expression.Unlike antigenic upregulation induced by the phorbol esters and ADMBwhich was inhibited by H-7, even the higher dose of H-7 (1.0 μg/ml) didnot inhibit the ability of IFN-β or IFN-γ to enhance BCA 225, HLA ClassII antigens and ICAM-1 expression in T47D cells.

To determine if H-7 could inhibit the ability of interferon to enhancethe synthesis or shedding of BCA 225 in T47D cells, cultures were grownfor 72 hr in the presence of 500 units/ml of IFN-β or 50 units/ml ofIFN-γ and in the presence or absence of 1 μg/ml of H-7 (Table 4). As hadbeen observed for interferon enhanced expression of BCA 225, H-7 did notinhibit the synthesis or shedding of BCA 225 induced in T47D cells byinterferon. These results demonstrate that the ability of TPA and MEZversus IFN-β and IFN-γ to upregulate the synthesis, expression andshedding of BCA 225 may occur by different mechanisms.

Effect of ADMB, TPA, MEZ and Interferon, Alone and in Combination withH-7, on the Steady-State Levels of HLA Class I Antigens, HLA ClassII-DRβ Antigen, ICAM-1 and c-erbB-2 in T47D Cells

To determine if the increase in HLA Class II-DR_(β) antigen, ICAM-1 andc-erbB-2 expression in T47D cells resulting from 72 hr ADMB, TPA, MEZ,IFN-α, IFN-β or IFN-γ treatment involved enhanced mRNA expression, thesteady-state levels of mRNA for the respective genes were determined(FIG. 3). In the case of HLA Class II-DR antigen, small increases inmRNA levels were observed in IFN-α (1.1-fold), IFN-β (1.2-fold), IFN-γ(1.25-fold) and MEZ (1.2-fold) treated T47D cells. ADMB and TPA did notincrease HLA Class II-DR_(β) antigen, RNA expression, although ADMB wasslightly more effective than MEZ in enhancing cell surface expression ofthis antigen in T47D cells (FIG. 2). MEZ and IFN-γ were the mosteffective enhancers of HLA Class II-DR_(β) antigen mRNA expression,whereas IFN-γ was more effective than MEZ in enhancing expression ofthis antigen in T47D cells (Tables 1 and 3). When cotreated with therespective antigenic modulating compound and H-7, only minimal changesin HLA Class II-DR_(β) antigen mRNA levels (<1.15-fold) were observed.These observations are in contrast to the HLA Class II-DR antigenicmodulation induced by these agents in T47D cells. As described above,H-7 effectively blocked ADMB, TPA and MEZ enhancement of HLA Class II-DRantigen expression in T47D cells (Table 3). In the case of HLA class Iantigen expression, mRNA levels were variably increased followingtreatment with IFN-α(1.9-fold), IFN-β (2.1-fold), IFN-γ(1.8-fold), TPA(1.3-fold) and MEZ (1.75-fold), whereas H-7 only marginally altered mRNAlevels (≦1.2-fold) for this antigen (Table 3). With respect to surfaceexpression, as observed with HLA Class II-DR antigen expression, H-7inhibited upregulation induced by ADMB, TPA or MEZ, but not upregulationinduced by the interferons (data not shown). These observations indicatethat cell surface expression changes in both HLA Class I antigens andHLA Class II-DR antigen in T47D cells is not a consequence of areduction in mRNA levels for these gene products. Further studies arerequired, however, to determine if the antigenic modulating agents,employed alone or in combination with H-7, modulate the rate of mRNAsynthesis and/or decay of mRNA synthesis for HLA Class II-DR or HLAClass I antigens in T47D cells.

ICAM-1 mRNA levels were increased a maximum of only 1.3-fold after 72 hrtreatment under the various experimental conditions and H-7 onlymodestly altered ICAM-1 expression (FIG. 3). In the case of c-erbB-2, amaximum increase of only 1.2-fold in the levels of mRNA were apparentafter 72 hr treatment with the various agents. Similarly, nodifferential change in c-erbB-2 mRNA was observed in T47D cells grown inthe presence of any of the antigenic modulating agents plus H-7. Theseresults again contrast those measuring surface expression of theseantigens. As demonstrated in Tables 1 and 3, H-7 partially inhibitedenhanced ICAM-1 expression (Table 3). Similarly, H-7 inhibited enhancedsurface expression of c-erbB-2 induced by ADMB, TPA and MEZ (FIG. 2 andTable 1), whereas it did not alter upregulation induced by theinterferons (data not shown). These results provide further support forthe lack of a direct correlation between the levels of ICAM-1 andc-erbB-2 mRNA and antigenic expression in cells treated with thecombination of ADMB, TPA, MEZ or interferon and H-7. Further studies arerequired, however, to determine if the antigenic modulating agents,employed alone or in combination with H-7, modulate the rate of mRNAsynthesis and/or decay of mRNA synthesis for ICAM-1 or c-erbB-2 in T47Dcells.

Effect of 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl Alcohol(ADMB), Mezerein (MEZ) and Recombinant Human Immune Interferon (IFN-1)on Antigen Expression in WiDr Human Colon Carcinoma Cells Monitored byFluorescence Activated Cell Sorter (FACS) Analysis

Cells were plated in 10 cm plates, media was changed 24 hours later withno addition (control) or addition of the indicated compounds and cellswere incubated at 37° C. for the indicated time (72 or 96 hours). Cellswere resuspended using 10× EDTA and antigen expression was determined asdescribed (Leon, Mesa-Tejada, Gutierrez, Estabrook, Greiner, Schlom &Fisher: Anticancer Res. 9:1639-1648, 1989; Leon, Gutierrez, Jiang,Estabrook, Waxman & Fisher: Cancer Immunol. Immmunother. 35:315-324,1992). The results of the experiments are presented in Table 5.

To summarize, Table 5 indicates: 1) class II HLA-DR antigens and thetumor associated antigen CA19.9 are elevated in WiDr human coloncarcinoma cells treated for 72 or 96 hours with ADMB, MEZ or IFN-γ; 2)the order of effectiveness in increasing Class II HLA-DR antigens inWiDr cells is IFN-γ>MEZ>ADMB (IFN-γ is approximately 3× more active thanADMB); 3) with all three immunomodulating agents (IFN-γ, MEZ and ADMB),96 hour treatment is more effective than 72 hours in inducing elevatedClass II HLA-DR and CA19.9 antigen expression; and 4) in the case of theTAA CA19.9, MEZ is more effective than IFN-γ or ADMB in increasingexpression after 96 hours treatment.

Conclusion

ADMB, as well as two documented immunomodulating agents (IFN-γ and MEZ),can increase the surface expression of both Class II HLA-DR and the TAACA19.9 in the human colon carcinoma cell line WiDr.

Effect of 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl Alcohol(ADMB), Mezerein (MEZ) and Recombinant Human Immune Interferon (IFN-y)on Antigen Expression in CBS Human Colon Carcinoma Cells Monitored byFluorescence Activated Cell Sorter (FACS) Analysis

Cells were plated in 10 cm plates, media was changed 24 hours later withno addition (control) or addition of the indicated compounds and cellswere incubated at 37° C. for 96 hours. Cells were resuspended using lOXEDTA and antigen expression was determined as described (Leon,Mesa-Tejada, Gutierrez, Estabrook, Breiner, Schlom & Fisher: AnticancerRes. 9:1639-1648, 1989; Leon, Gutierrez, Jiang, Estabrook, Waxman &Fisher: Cancer Immuno. Immunother. 35:315-324 1992). The results of theexperiments are presented in Table 6.

To summarize, Table 6 indicates: 1) Class II HLA-DR antigens and thetumor associated antigen CA19.9 are elevated in CBS human coloncarcinoma cells treated for 96 hours with ADMB, MEZ or IFN-γ; 2) therelative order of effectiveness in increasing Class II HLA-DR antigensin CBS cells is IFN-γ>MEZ>ADMB. (IFN-γ is approximately 2× more activethan ADMB); and 3) ADMB is more effective than MEZ in enhancing CA19.9TAA expression, but less effective than IFN-γ (Rank order ofeffectiveness IFN-γ>ADMB>MEZ).

Conclusion

ADMB, as well as two documented immunomodulating agents (IFN-γ and MEZ),can increase the surface expression of both Class II HLA-DR and the TAACa19.9 in the human colon carcinoma cell line CBS.

Summary Conclusion

ADMB can augment Class II HLA-DR antigen and CA19.9 TAA expression inhuman colon carcinoma cells (WiDr and CBS).

The data in Tables 5 and 6 demonstrate that ADMB can augment both ClassII HLA-DR and TAA expression in two human colon carcinoma cell lines. Itappears, therefore, that ADMB, and mechanistically similar compounds,should find utility in increasing HLA and TAA expression in both breastand colon carcinoma cells. Studies are continuing to determine if ADMBcan stimulate antigenic expression in additional carcinomas.

EXPERIMENTAL DISCUSSION

Among the diversity of interferon effects on target cells, recentinvestigations have focused on the ability of these bioresponsemodulators to enhance the expression of both histocompatibility antigensand TAAs in tumor cells (for review see [1,9,23,25,26]) These studiesindicate that interferon may prove valuable in altering the phenotype oftumor cells rendering them more accessible to monoclonal antibodytargeting [13,21,36,42]. A frequent observation is that interferonfunctions predominantly as an enhancer of antigenic expression, ratherthan an inducer of de novo expression of specific antigens[9,15,16,25,31]. At the present time, little information is available onthe biochemical mechanisms underlying interferon upregulation ofantigenic expression. Studies comparing the protein synthesisrequirements for antigenic modulation induced by types I (IFN-α/β) andtype II (IFN-γ) interferon in human melanoma cells suggest thatdifferent biochemical pathways mediate up-regulation of both majorhistocompatibility complex (MHC) and non-MHC encoded glycoproteinsinduced by these compounds [17]. IFN-γ enhancement of antigen expressiondepends on continued protein synthesis, whereas the modulatory effect ofIFN-α and IFN-β can occur in the presence of the protein synthesisinhibitor cycloheximide. Numerous studies have also indicated that typesI and II interferons can differ in their effects on TAA expression inthe same tumor cell (for review see [1,23,25,48]). Both the absolutelevel of antigenic modulation induced by different interferons, as wellas the type of effect elicited in specific target cells, i.e. eitherstimulatory or inhibitory, has been shown to vary (for review see[1,23]). In the present study applicants have addressed the potentialrelationship between PKC activation and antigenic modulation induced byrecombinant IFN-β and IFN-γ in the human breast carcinoma cell lineT47D. Since both TPA and MEZ can augment the expression of the samehistocompatibility antigens and TAAs in T47D cells as recombinantinterferons and these agents appear to work directly via activation ofPKC (for review see [43,44]), applicants have also incorporated theseagents in our studies.

The enhanced cellular antigenic expression induced in T47D cells by TPA,MEZ and ADMB was eliminated by simultaneous incubation with thePKC-inhibitor H-7. Similarly, the ability of TPA and MEZ to enhance thesynthesis and shedding of the TAA BCA 225 was also blocked by H-7. Incontrast, ADMB failed to induce increased shedding of BCA 225 by T47Dcells, whereas it was similarly active as TPA in enhancing BCA 225synthesis and expression (Table 2). These observations suggest that ADMBcan differentially modify the antigenic phenotype in T47D cells incomparison with TPA and MEZ, i.e. it can augment synthesis andexpression without enhancing shedding of specific TAAs. Interestingly,ADMB was found to induce HLA Class II antigens in a similar manner asTPA and MEZ in T47D cells. HLA Class II antigens have been shown to beinvolved in the differentiation of mammary epithelium [11] and they playa critical role in antigen presentation to T-cells [35], the transportof key intracellular peptides to the extracellular milieu [57] andrecruitment of lymphoid cells to tumor cells [11,35,59]. The ability ofADMB to enhance both HLA Class II antigens and TAA expression on T47Dcells could have implications with respect to the induction of an immuneresponse to this tumor in vivo. A severe limitation preventing the useof TPA or MEZ as potential immunomodulators in humans is there welldocumented tumor promoting activity in the mouse skin two-stagecarcinogenesis assay (for review see [51-52]). At the present time, ADMBhas not been tested for in vivo toxicity, tumor promoting activityand/or in vivo immunomodulatory properties. However, it this compoundcan pass this scrutiny, it could prove useful as an antigenic modulatingagent in situations where increased surface expression without aconcomitant increase in TAA shedding is desired.

Previous studies have indicated a possible involvement of activation ofPKC in the early events associated with IFN-α action in specific targetcells [2,6,7,46,50,60-62]. Although not studied as extensively, anassociation between PKC activity and both IFN-β- and IFN-γ-inducedcellular changes has also been suggested [8,29,30,45,47]. In addition, adifferential role for PKC in IFN-α/β versus IFN-γ induced cellular andgene expression changes in the same target cell has also been observed[6,37,47]. Both IFN-β and IFN-γ produced similar antigenic changes inT47D cells as TPA and MEZ, including enhancing the shedding of BCA 225.However, up-regulation of antigen expression and increased sheddinginduced by IFN-β and IFN-γ was not inhibited by H-7 (Table IV). IFN-αwas less effective than either IFN-β or IFN-γ in modifying the antigenicphenotype of T47D cells and its activity also was not blocked by H-7(data not shown). These results suggest that the mechanism by whichinterferons modulate antigen expression in T47D cells occurs by aPKC-independent pathway. A similar dissociation between PKC activationand the ability of IFN-γ and TPA to induce specific antigenic expressionchanges in human keratinocyte cultures has recently been reported [24].IFN-γ and TPA both enhanced ICAM-1 expression in human keratinocytecultures and the enhancement effect of TPA, but not that of IFN-γ, wasinhibited by H-7. Similarly, only IFN-γ induced HLA Class II-DR antigenexpression in human keratinocytes and H-7 also failed to block thisinduction. Koide et al. [34] demonstrated that IFN-γ induction of HLAClass II-DR antigen expression in HL-60 cells also was not modified byH-7. In contrast, W7 (a calmodulin antagonist) blocked IFN-γ-inductionof HLA Class II-DR expression in HL-60 cells supporting a possibleinvolvement of calcium/calmodulin in antigenic modulation in this cellline. Similarly, treatment of murine macrophages with IFN-γ resulted inthe induction of both increased mRNA and MHC I-A_(β) antigen expression,and both of these parameters were unaltered in the presence of H-7 [5].W-7 did, however, modify the MHC I-A_(β) antigen MRNA induction processelicited by IFN-γ treatment in murine macrophages. In the case of thehuman melanoma cell line H0-1, the enhanced expression of HLA Class Iantigens, HLA Class II antigens and ICAM-1 induced by IFN-γ was againonly marginally affected by H-7 [26] . Since upregulation of antigenexpression in T47D cells induced by PKC activators such as TPA, MEZ andADMB are inhibited by H-7, whereas similar changes induced by theinterferons are not blocked by H-7, these results further indicate thatthe mechanism controlling antigenic modulation in specific cell culturesis dependent on the specific inducer employed and antigenic modulationcan occur by both a PKC-independent and a PKC-dependent pathway.

The mechanism by which ADMB and MEZ versus IFN-γ enhance the expressionof specific cellular antigens and TAAs in T47D is not presently known.Analysis of steady state mRNA levels of HLA Class I, HLA ClassII-DR_(β), ICAM-1 and c-erbB-2 in cells treated with these differentcompounds indicated various levels of modulation which did not correlatedirectly with the relative level of change in surface expression ofthese antigens. Similarly, H-7 did not significantly alter the level ofmRNA for the various antigens under any of the experimental conditions.These results suggest that the ability of H-7 to modulate the antigenicenhancing properties of ADMB, TPA, MEZ and the interferons may occur ata posttranscriptional level. Alternatively, these agents may modifyantigenic expression by altering the rate of mRNA transcription and/ormRNA stability. In the case of BCA 225, H-7 may exert its suppressiveeffect on ADMB-, TPA-, and MEZ-induced increases in surface expressionby inhibiting the ability of these compounds to enhance the synthesis ofthis TAA in T47D cells. Alternatively, H-7 might block antigenicupregulation in ADMB, TPA and MEZ treated cells by preventing thenecessary biochemical alterations responsible for the insertion of thevarious antigens into the cell membrane in a form recognized by themonoclonal antibodies employed. Further studies are clearly required todetermine the mechanism by which specific antigenic modulatorsupregulate antigen expression and the mechanism by which H-7 selectivelyinhibits this process in cells treated with ADMB, TPA or MEZ. Thepresent model system should prove useful in determining the biochemicalmechanism(s) underlying antigenic upregulation in response to diversetransmembrane signalling agents. With this information it may bepossible in the future to design strategies and molecules specificallytailored to alter the antigenic phenotype of tumor cells making themmore accessible to monoclonal antibody targeted therapeutic approaches.

TABLE 1 Effect of the PKC Inhibitor H-7 on Upregulation of HLA Class IIantigens, c-erbB-2 and ICAM-1 by TPA and MEZ in T47D Human BreastCarcinoma Cells. Antigenic Expression (MFI) Experimental HLA ClassConditions^(a) II c-erbB-2 ICAM-1 Control 3,410 7,144 20,975 H-7 4,5637,045 21,538 (1.3)^(b) (1.0) (1.0) TPA 9,984 16,100 63,175 (2.9) (2.3)(3.0) TPA + H-7 4,778 8,385 47,542 (1.4) (1.2) (2.3) MEZ 10,260 18,38585,745 (3.0) (2.6) (4.1) MEZ + H-7 3,654 Not 39,486 (1.1) Detected (1.9)^(a)T47D cells were grown for 72 hr in 0.1 μg/ml TPA or 0.1 μg/ml MEZ,in the presence or absence of 0.1 μg/ml H-7. Cells were resuspended,incubated with monoclonal antibodies specific for HLA Class II antigens,c-erbB-2 or ICAM-1 and anti-mouse FITC secondary antibody. Cells werethen analyzed by flow cytometry using a FACStar (Beckon Dickinson,Mountain View, CA) and antigenic expression is expressed as meanfluorescence intensity (MFI) units. Further details can be found in #“Detailed Description of the Invention.” ^(b)Numbers in brackets reflectthe level of upregulation versus untreated control cells (equivalent to1.0)

TABLE 2 Effect of the PKC Inhibitor H-7 on the Enhanced Synthesis,Expression and Shedding of BCA 225 Induced by TPA, MEZ and ADMB in T47DHuman Breast Carcinoma Cells. BCA 225 BCA 225 BCA 225 Shedding^(d)Experimental Synthesis^(b) Expression^(c) (ng/ml/10⁶ Conditions^(a)(ng/mg protein) (MFI) cells) Control 164 6,426 28 H-7 142 (0.9)^(e)5,428 (0.8) 23 (0.8) TPA 394 (2.4) 11,696 (1.8) 84 (3.0) TPA + H-7 191(1.2) 7,434 (1.2) 35 (1.3) MEZ 507 (3.1) 16,065 (2.5) 147 (5.3) MEZ +H-7 225 (1.4) 7,068 (1.1) 39 (1.4) ADMB 299 (1.8) 12,825 (2.0) 26 (0.9)ADMB + H-7 178 (1.1) 7,018 (1.1) 34 (1.2) ^(a)T47D cells were incubatedfor 72 hr in the presence of 0.1 μg/ml TPA, 0.1 μg/ml MEZ or 0.25 μg/mlADMB, in the presence or absence of 0.1 μg/ml H-7. ^(b)Cell lysates wereprepared and BCA 225 levels were determined by double-determinant BCA225 ELISA assay as described in “Detailed Description of the Invention.”^(c)Membrane expression of BCA 225 was determined by flow cytometryusing a FACStar (Beckon Dickinson, Mountain View, CA) with CU18monoclonal antibodies as described in “Detailed Description of theInvention.” The results are expressed as mean fluorescence intensity(MFI) units. ^(d)The shedding of BCA 225 into the culture medium wascalculated using a double-determinant BCA 225 ELISA procedure asdescribed in “Detailed Description of the Invention.” ^(e)Values inbrackets reflect the level of enhancement in BCA 225 relative to control(equivalent to 1.0)

TABLE 3 Effect of the PKC Inhibitor H-7 on Upregulation of HLA Class IIantigens and ICAM-1 by IFN-β and IFN-γ in T47D Human Breast CarcinomaCells. Experimental Antigenic Expression (MFI) Conditions^(a) HLA ClassII ICAM-1 Control 2,919 54,536 H-7 3,490 (1.2)^(b) 54,080 (1.0) IFN-β31,413 (10.8) 97,013 (1.8) IFN-β + H-7 32,190 (11.1) 108,902 (2.0) IFN-γ202,102 (69.2) 191,490 (3.5) IFN-γ + H-7 210,812 (75.2) 188,544 (3.5)^(a)T47D cells were incubated for 72 in the presence of 500 units/mlIFN-β or 50 units/ml IFN-γ, in the presence or absence of 1.0 μg/ml ofH-7. Cells were then incubated with Monoclonal Antibodies specific forHLA Class II antigens or ICAM-1 followed by fluorescinated anti-mouseIgG antibody and then analyzed by flow cytometry with a Coulter Epics IVFACS (Coulter Electronics, Hialeah, FL.) as described in “DetailedDescription of the Invention.” Results are expressed as mean #fluorescence intensity (MFI) units. ^(b)Values in brackets indicate therelative increase in expression versus untreated controls (equivalent to1.0)

TABLE 4 Effect of the PKC Inhibitor H-7 on the Enhanced Synthesis,Expression and Shedding of BCA 225 Induced by IFN-β and IFN-γ in T47DHuman Breast Carcinoma Cells. BCA 225 BCA 225 BCA 225 Shedding^(d)Experimental Synthesis^(b) Expression^(c) (ng/ml/109⁶ Conditions^(a)(ng/mg/protein) (MFI) cells) Control 223 2,816 47 H-7 230 (1.0)^(e)2,560 (0.9) 38 (0.8) IFN-β 374 (1.7) 5,716 (2.0) 69 (1.5) IFN-β + H-7361 (1.6) 5,714 (2.0) 66 (1.4) IFN-γ 722 (3.2) 9,069 (3.2) 64 (1.4)IFN-γ + H-7 694 (3.1) 8,177 (2.9) 64 (1.4) ^(a)T47D cells were incubatedfor 72 hr with 500 units/ml IFN-β or 50 units/ml of IFN-γ, in thepresence or absence of 1.0 μg/ml of H-7. ^(b)BCA 225 synthesis wascalculated by ELISA using cell lysates as described in “DetailedDescription of the Invention.” ^(c)BCA 225 cell surface expression wasmeasured by flow cytometry using a FACStar (Beckon Dickinson, MountainView, CA) as described in “Detailed Description of the Invention.”Results are expressed as mean fluorescence intensity (MFI) units.^(d)Shedding of BCA 225 into the culture medium was measured by ELISA asdescribed in “Detailed Description of the Invention.” ^(e)Values inbrackets indicate the relative change in BCA 225 in comparison withcontrols (equivalent to 1.0).

TABLE 5 Effect of 3-(N-acetylamino)-5-(N-decyl-N- methylamino)-benzylalcohol (ADMB), Mezerein (MEZ) and Recombinant Human Immune Interferon(IFN-γ) on Antigen Expression in WIDr Human Colon Carcinoma CellsMonitored by Fluorescence Activated Cell Sorter (FACS) AnalysisMonoclonal Antibody Binding (MFI)^(b) Experimental MoAb L243 MoAb B67.4Conditions^(a) MoAb K56 (HLA-DR) (CA19.9) 72 Hour Treatment Control 840342 1260 ADMB (0.01 μg/ml) 844 390 1136 ADMB (0.1 μg/ml) 715 317 1172ADMB (1.0 μg/ml) 879 486* 1400* MEZ (100 ng/ml) 964* 1120* 1914* IFN-γ(100 U/ml) 1046* 1724* 2271* 96 Hour Treatment Control 1018 492 1475ADMB (0.01 μg/ml) 827 434 1512 ADMB (0.1 μg/ml) 1116 615* 2028* ADMB(1.0 μg/ml) 1008 806* 2417* MEZ (100 ng/ml) 1104 2129* 4784* IFN-γ (100U/ml) 1021 2376* 3191* ^(a)Cells were plated in 10 cm plates, media waschanged 24 hours later with no addition (control) or addition of theindicated compounds and cells were incubated at 37° C. for the indicatedtime (72 or 96 hours). Cells were resuspended using 10X EDTA and antigenexpression was determined as described (Leon, Mesa-Tejada, Gutierrez,Estabrook, Greiner, Schlom & Fisher: Anticancer Res. 9: 1639-1648, 1989;Leon, Gutierrez, Jiang, Estabrook, Waxman & Fisher: Cancer Immunol.Immmunother. # 35:315-324, 1992) ^(b)Values presented represent meanfluorescence intensity units (MFI). MFI = (mean channel fluorescence influorescence positive antibody-binding cells × % of Fluorescencepositive antibody-binding cells) − (mean channel fluorescence ofunstained cells × % of fluorescence positive cells in the unstainedpopulation). # The data presented reflects the average of duplicatesamples per experimental point which varied by ≦ 10%. Antibodies usedincluded: MoAb K56, MoAb L243 (recognizes HLA-DR) and MoAb B67.4(recognizes the tumor associated antigen, CA19.9). *Enhanced antigenexpression.

TABLE 6 Effect of 3-(N-acetylamino)-5-(N-decyl-N- methylamino)-benzylalcohol (ADMB), Mezerein (MEZ) and Recombinant Human Immune Interferon(IFN-γ) on Antigen Expression in WIDr Human Colon Carcinoma CellsMonitored by Fluorescence Activated Cell Sorter (FACS) AnalysisMonoclonal Antibody Binding (MFI)^(b) Experimental MoAb L243 MoAb B67.4Conditions^(a) MoAb K56 (HLA-DR) (CA19.9) Control 2034 3941 1509 ADMB(0.01 μg/ml) 2126 4112 3562* ADMB (0.1 μg/ml) 1715 3328 3473* ADMB (1.0μg/ml) 2420 6870* 3962* MEZ (100 ng/ml) 3002* 9726* 3288* IFN-γ (100U/ml) 3126* 14972* 4970* ^(a)Cells were plated in 10 cm plates, mediawas changed 24 hours later with no addition (control) or addition of theindicated compounds and cells were incubated at 37° C. for 96 hours.Cells were resuspended using 10X EDTA and antigen expression wasdetermined as described (Leon, Mesa-Tejada, Gutierrez, Estabrook,Greiner, Schlom & Fisher: Anticancer Res. 9:1639-1648, 1989; Leon,Gutierrez, Jiang, Estabrook, Waxman & Fisher: Cancer Immunol.Immunother. 35:315-324 1992). ^(b)Values presented represent meanfluorescence intensity units (MFI). MFI = (mean channel fluorescence influorescence positive antibody-binding cells C % of Fluorescencepositive antibody-binding cells) − (mean channel fluorescence ofunstained cells X % of fluorescence positive cells in the unstainedpopulation). The data presented reflects the average of duplicatesamples per experimental point which varied by ≦ 10% Antibodies usedincluded: MoAb K56, MoAb L243 (recognizes # HLA-DR) and MoAb B67.4(recognizes the tumor associated antigen, CA19.9) *Enhanced antigenexpression.

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10. Fisher, P. B., Schachter, D., Mufson, R. A, Huberman, E. The role ofmembrane lipid dynamics and translocation of protein kinase C in theinduction of differentiation in human promyelocytic leukemic cells. In:Kabara JJ (ed.) Pharmacological effect of lipids: III. role of lipids incancer research, The American Oil Chemists' Society, Champaign, Ill.,pp. 69-89 (1989).

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12. Fort, P., Marty, L., Piechaczyk, M., Sabrouty, S. E., Dani, C.,Jeanteur, P., Blanchard, J. M. Various rat adult tissues express onlyone major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenasemultigenic family, Nucleic Acids Res., 13: 1431 (1985).

13. Fuith, L. C., Marth, C., Muller-Holzner, E., Zechmann, W.,Daxenbichler, G. Enhancement of CA 125 expression by interferon-gamma inovarian carcinoma xenografts, J. Tumor Marker Oncology, 6: 85 (1991).

14. Giacomini, P., Aguzzi, A., Pestka, S., Fisher P. B., Ferrone, S.Modulation by recombinant DNA leukocyte (α) and fibroblast (β)interferons of the expression and shedding of HLA and tumor associatedantigens by human melanoma cells, J. Immunol., 133: 1649 (1984).

15. Giacomini, P., Fisher, P. B., Duigou, G. J., Gambari, R., Natali, P.G. Regulation of class II MHC gene expression by interferons: insightsinto the mechanism of action of interferon, Anticancer Res., 8: 1153(1988).

16. Giacomini, P., Fraioli, R., Nistico, P., Tecce, R., Nicotra, M. R.,Di Filippo, F., Fisher, P. B., Natali, P. G. Modulation of the antigenicphenotype of early-passage human melanoma cells derived from multipleautologous metastases by recombinant human leukocyte, fibroblast andimmune interferon, Int. J. Cancer, 46: 539 (1990).

17. Giacomini, P., Tecce, R., Sacchi, A., Fisher, P. B., Natali, P. G.Recombinant human IFN-γ, but not IFN-α or IFN-β, enhances MHC and nonMHC encoded glycoproteins by a protein synthesis dependent mechanism, J.Immunol., 140:3073 (1988).

18. Graham, G. M., Guarini, L., Moulton, T. A., Datta, S., Ferrone, S.,Giacomini, P., Kerbel, R. S., Fisher, P. B. Potentiation of growthsuppression and modulation of the antigenic phenotype in human melanomacells by the combination of recombinant human fibroblast and immuneinterferons, Cancer Immunol. Immunother., 32: 382 (1991).

19. Gray, P. W., Leung, B., Pennica, D., Yelverton, E., Najarian, R.,Simonsen, C. C., Derynk, R., Sherwood, P. J., Wallace, D. M., Berger, S.L., Levinson, A. D., Goeddel, D. V. Expression of human immuneinterferon cDNA in E. coli and monkey cells, Nature, 295:502 (1982).

20. Greiner, J. W., Fisher, P. B., Pestka, S., Schlom, J. Differentialeffects of recombinant human leukocyte interferons on cell surfaceantigen expression, Cancer Res., 46:4894 (1986).

21. Greiner, J. W., Guadagni, F., Noguchi, P., Pestka, S., Colcher, D.,Fisher, P. B., Schlom. J. Use of recombinant interferon to enhancemonoclonal antibody-targeting of carcinoma lesions in vivo, Science,235: 895 (1987).

22. Greiner, J. W., Hand, P. H., Noguchi, P., Fisher, P. B., Pestka, S.,Schlom, J. Enhanced expression of surface tumor-associated antigens onhuman breast and colon tumor cells after recombinant leukocyteα-interferon treatment, Cancer Res., 44: 3208 (1984).

23. Greiner, J. W., Schlom, J., Pestka, S., Langer, J. A., Giacomini,P., Kusama, M., Ferrone, S., Fisher, P. B. Modulation of tumorassociated antigen expression and shedding by recombinant humanleukocyte and fibroblast interferons, Pharmacol. Therapeut., 31:209(1985).

24. Griffiths, C. E. M., Esmann, J., Fisher, G. J., Voorhees, J. J.,Nickoloff, B. J. Differential modulation of keratinocyte intercellularadhesion molecule-1 expression by gamma interferon and phorbol ester:evidence for involvement of protein kinase C signal transduction,British J. Dermatology, 122:333 (1990).

25. Guadagni, F., Kantor, J., Schlom, J., Greiner, J. W. Regulation oftumor antigen expression by recombinant interferons, In: Fisher, P. B.(ed.), Mechanisms of differentiation: II. modulation of differentiationby exogenous agents, CRC Press, Boca Raton, pp. 57-80 (1990).

26. Guarini, L., Graham, G. M., Jiang, H., Ferrone, S., Zucker, S.,Fisher, P. B. Modulation of the anitigenic phenotype of human melanomacells by differentiation-inducing and growth-suppressing agents, PigmentCell Res. Suppl., 2:123-131 (1992).

27. Guarini, L., Temponi, M., Bruce, J. N., Bollon, A. P., Duigou, G.J., Moulton, T. A., Ferrone, S., Fisher, P. B. Expression and modulationby cytokines of the intercellular adhesion molecule-1 (ICAM-1) in humancentral nervous system tumor cell cultures, Int. J. Cancer, 46:1041(1990).

28. Guarini, L., Temponi, M., Edwalds, G. M., Vita, J. R., Fisher, P.B., Ferrone, S. In vitro differentiation and antigenic changes in humanmelanoma cell lines, Cancer Immunol. Immunother., 30:262 (1989).

29. Hamilton, T. A., Becton, D. L., Somer, S. D., Gray, P. W., Adams, D.O. Inteferon-γ modulates protein kinase C activity in murine peritonealmacrophages, J. Biol. Chem., 260:1378 (1985).

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31. Kantor, J., Tran R., Greiner, J., Pestka, S., Fisher, P. B., Schlom,J. Modulation of carcinoembryonic antigen messenger RNA levels in humancolon carcinoma cells by recombinant human γ-interferon, Cancer Res.,49:2651 (1989).

32. Keydar, I., Ohno, T., Nayak, T., Sweet, R., Simoni, F., Weiss, F.,Karby, S., Mesa-Tejada, R., Spiegelman, S. Properties of retrovirus-likeparticles produced by a human breast carcinoma cell line: immunologicrelationship with mouse mammary tumor virus proteins, Proc. Natl. Acad.Sci., 81:4188 (1984).

33. Klareskog, L., Forsum, U., Peterson, P. A. Hormonal regulations ofthe expression of Ia antigens on mammary gland epithelium, Eur. J.Immunol., 10: 958 (1985).

34. Koide, Y., Ina, Y., Nezu, N., Yoshida, T. O. Calcium influx andCa²⁺-calmodulin complex are involved in interferon-γ-induced expressionof HLA Class II molecules on HL-60 cells, Proc. Natl. Acad. Sci.,85:3120 (1988)

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36. Leon, J. A., Mesa-Tejada, R., Gutierrez, M. C., Estabrook, A.,Greiner, J. W., Schlom, J., Fisher, P. B. Increased surface expressionand shedding of tumor associated antigens by human breast carcinomacells treated with recombinant human interferons or phorbol ester tumorpromoters, Anticancer Res., 9:1639 (1989).

37. Lew, D. J., Decker, T., Darnell, J. E., Jr. Alpha interferon andgamma interferon stimulate transcription of a single gene throughdifferent signal transduction pathways, Mol. Cell. Biol., 9:5405 (1989).

38. Maio, M., Gulwani, B., Langer, J. A., Kerbel, R. S., Duigou, G. J.,Fisher, P. B., Ferrone, S. Modulation by interferons of HLA antigens,high molecular weight-melanoma associated antigens and intercellularadhesion molecule-1 expression by cultured melanoma cells with differentmetastatic potential, Cancer Res., 49:2980 (1989).

39. Mark, D. V., Lu, S. D., Creasey, A., Yamamoto, R., Lin, L.Site-specific mutagenesis of the human fibroblast interferon gene, Proc.Natl. Acad. Sci. USA, 81:5662 (1984).

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47. Razdioch, D., Varesio, L. Protein kinase C inhibitors block theactivation of macrophages by IFN-β but not by IFN-γ, J. Immunol.,140:1259 (1988).

48. Reddy, P. G., Graham, G. M., Datta, S., Guarini, L., Moulton, T. A.,Jiang, H., Gottesman, M. M., Ferrone, S., Fisher, P. B. Effect ofrecombinant fibroblast interferon and recombinant immune interferon ongrowth and the antigenic phenotype of multidrug-resistant humanglioblastoma multiforme cells, J. Natl. Cancer Inst., 83:1307 (1991).

49. Rehberg, E., Kelder, B., Hoal, E. G., Pestka, S. Specific molecularactivities of recombinant and hybrid leukocyte interferons, J. BiolChem., 257:11497 (1982).

50. Reich, N. C., Pfeffer, L. M. Evidence for involvement of proteinkinase C in the cellular response to interferon α, Proc. Natl. Acad.Sci. USA, 87:8761 (1990).

51. Slaga, T. J., Fischer, S. M., Nelson, K., Gleason, G. L. Studies onthe mechanism of skin tumor promotion: evidence for several stages inpromotion, Proc. Natl. Acad. Sci. USA, 77:3659 (1980).

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What is claimed is:
 1. A method of detecting tumor cells comprisingcontacting tumor cells with an effective amount of a protein kinase Cactivator for the upregulation of expression of cell antigens of tumorcells, without inducing antigen shedding, and detecting the presence ofsaid antigen.
 2. The method of claim 1, wherein the protein kinase Cactivator is a synthetic protein kinase C activator.
 3. The method ofclaim 2, wherein the protein kinase C activator is3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol.
 4. Themethod of claim 1, wherein the cell antigen is selected from a groupconsisting of a tumor associated antigen, a cell surface antigen, and ahistocompatibility antigen.
 5. The method of claim 4, wherein the tumorof the tumor associated antigen is a breast carcinoma.
 6. The method ofclaim 4, wherein the tumor of the tumor associated antigen is a coloncarcinoma.
 7. The method of claim 5, wherein the tumor associatedantigen is selected from a group consisting of BCA 225, carcinoembryonicantigen, and c-erb B2.
 8. The method of claim 5, wherein the antigen isthe cell surface antigen intercellular adhesion molecule-1.
 9. Themethod of claim 3, wherein the effective amount is from about 0.01 μg/mlto about 10 μg/ml.
 10. The method of claim 4, wherein the antigen isassociated with a breast or colon carcinoma and is selected from a groupconsisting of a histocompatability antigen, Class II HLA-DR, and CA19.9.